Regulation of Fibroblast Growth Factor-23 Signaling by Klotho

The aging suppressor gene Klotho encodes a single-pass transmembrane protein. Klotho-deficient mice exhibit a variety of aging-like phenotypes, many of which are similar to those observed in fibroblast growth factor-23 (FGF23)-deficient mice. To test the possibility that Klotho and FGF23 may function in a common signal transduction pathway(s), we investigated whether Klotho is involved in FGF signaling. Here we show that Klotho protein directly binds to multiple FGF receptors (FGFRs). The Klotho-FGFR complex binds to FGF23 with higher affinity than FGFR or Klotho alone. In addition, Klotho significantly enhanced the ability of FGF23 to induce phosphorylation of FGF receptor substrate and ERK in various types of cells. Thus, Klotho functions as a cofactor essential for activation of FGF signaling by FGF23. The aging suppressor gene Klotho encodes a single-pass transmembrane protein. Klotho-deficient mice exhibit a variety of aging-like phenotypes, many of which are similar to those observed in fibroblast growth factor-23 (FGF23)-deficient mice. To test the possibility that Klotho and FGF23 may function in a common signal transduction pathway(s), we investigated whether Klotho is involved in FGF signaling. Here we show that Klotho protein directly binds to multiple FGF receptors (FGFRs). The Klotho-FGFR complex binds to FGF23 with higher affinity than FGFR or Klotho alone. In addition, Klotho significantly enhanced the ability of FGF23 to induce phosphorylation of FGF receptor substrate and ERK in various types of cells. Thus, Klotho functions as a cofactor essential for activation of FGF signaling by FGF23. The Klotho gene encodes a 130-kDa single-pass transmembrane protein with a short cytoplasmic domain (10 amino acids) and is expressed predominantly in the kidney. Mice carrying a loss-of-function mutation in the Klotho gene develop a syndrome resembling human aging, including shortened life span, skin atrophy, muscle atrophy, osteoporosis, arteriosclerosis, and pulmonary emphysema (1Kuro-o M. Matsumura Y. Aizawa H. Kawaguchi H. Suga T. Utsugi T. Ohyama Y. Kurabayashi M. Kaname T. Kume E. Iwasaki H. Iida A. Shiraki-Iida T. Nishikawa S. Nagai R. Nabeshima Y. Nature. 1997; 390: 45-51Crossref PubMed Scopus (2811) Google Scholar). Conversely, overexpression of the Klotho gene extends the life span and increases resistance to oxidative stress in mice (2Kurosu H. Yamamoto M. Clark J.D. Pastor J.V. Nandi A. Gurnani P. McGuinness O.P. Chikuda H. Yamaguchi M. Kawaguchi H. Shimomura I. Takayama Y. Herz J. Kahn C.R. Rosenblatt K.P. Kuro-o M. Science. 2005; 309: 1829-1833Crossref PubMed Scopus (1397) Google Scholar, 3Yamamoto M. Clark J.D. Pastor J.V. Gurnani P. Nandi A. Kurosu H. Miyoshi M. Ogawa Y. Castrillon D.H. Rosenblatt K.P. Kuro-o M. J. Biol. Chem. 2005; 280: 38029-38034Abstract Full Text Full Text PDF PubMed Scopus (528) Google Scholar, 4Ikushima M. Rakugi H. Ishikawa K. Maekawa Y. Yamamoto K. Ohta J. Chihara Y. Kida I. Ogihara T. Biochem. Biophys. Res. Commun. 2006; 339: 827-832Crossref PubMed Scopus (188) Google Scholar). These observations suggest that the Klotho gene functions as an aging suppressor gene. The extracellular domain of Klotho protein is shed and secreted in the blood (2Kurosu H. Yamamoto M. Clark J.D. Pastor J.V. Nandi A. Gurnani P. McGuinness O.P. Chikuda H. Yamaguchi M. Kawaguchi H. Shimomura I. Takayama Y. Herz J. Kahn C.R. Rosenblatt K.P. Kuro-o M. Science. 2005; 309: 1829-1833Crossref PubMed Scopus (1397) Google Scholar, 5Imura A. Iwano A. Tohyama O. Tsuji Y. Nozaki K. Hashimoto N. Fujimori T. Nabeshima Y. FEBS Lett. 2004; 565: 143-147Crossref PubMed Scopus (457) Google Scholar), potentially functioning as a humoral factor that signals suppression of intracellular insulin/IGF1 signaling, which partly contributes to its anti-aging properties (2Kurosu H. Yamamoto M. Clark J.D. Pastor J.V. Nandi A. Gurnani P. McGuinness O.P. Chikuda H. Yamaguchi M. Kawaguchi H. Shimomura I. Takayama Y. Herz J. Kahn C.R. Rosenblatt K.P. Kuro-o M. Science. 2005; 309: 1829-1833Crossref PubMed Scopus (1397) Google Scholar). However, a signaling pathway(s) directly activated by Klotho protein, including the identity of the Klotho receptor, has not been determined. The function of the transmembrane form of Klotho protein also remains to be determined. Fibroblast growth factor-23 (FGF23) 2The abbreviations used are: FGF, fibroblast growth factor; FGFR, fibroblast growth factor receptor; Ig-like, immunoglobulin-like; TBS, Tris-buffered saline; CHO, Chinese hamster ovary; GFP, green fluorescent protein; m.o.i., multiplicity of infection; ERK, extracellular signal-regulated kinase; MAP, mitogen-activated protein. was originally identified as a gene mutated in patients with autosomal dominant hypophosphatemic rickets (6The ADHR Consortium Nat. Genet. 2000; 26: 345-348Crossref PubMed Scopus (1281) Google Scholar), where mutations in the FGF23 gene conferred resistance to inactivation by protease cleavage, resulting in elevated serum levels of FGF23 (7White K.E. Carn G. Lorenz-Depiereux B. Benet-Pages A. Strom T.M. Econs M.J. Kidney Int. 2001; 60: 2079-2086Abstract Full Text Full Text PDF PubMed Scopus (440) Google Scholar, 8Liu S. Guo R. Simpson L.G. Xiao Z.S. Burnham C.E. Quarles L.D. J. Biol. 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FGF23 inhibits phosphate transport in renal proximal tubular cells and in proximal tubules perfused in vitro (13Baum M. Schiavi S. Dwarakanath V. Quigley R. Kidney Int. 2005; 68: 1148-1153Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). Consistent with these findings, mice defective in FGF23 expression show increased renal phosphate reabsorption and hyperphosphatemia (14Shimada T. Kakitani M. Yamazaki Y. Hasegawa H. Takeuchi Y. Fujita T. Fukumoto S. Tomizuka K. Yamashita T. J. Clin. Invest. 2004; 113: 561-568Crossref PubMed Scopus (1273) Google Scholar). Although FGF23 binds to multiple FGF receptors (FGFRs) (15Yu X. Ibrahimi O.A. Goetz R. Zhang F. Davis S.I. Garringer H.J. Linhardt R.J. Ornitz D.M. Mohammadi M. White K.E. Endocrinology. 2005; 146: 4647-4656Crossref PubMed Scopus (177) Google Scholar), it has modest receptor affinity (KD = 200–700 nm) and often requires cofactors such as heparin or glycosaminoglycan (15Yu X. Ibrahimi O.A. Goetz R. Zhang F. Davis S.I. Garringer H.J. Linhardt R.J. Ornitz D.M. Mohammadi M. White K.E. Endocrinology. 2005; 146: 4647-4656Crossref PubMed Scopus (177) Google Scholar, 16Yamashita T. Konishi M. Miyake A. Inui K. Itoh N. J. Biol. Chem. 2002; 277: 28265-28270Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar) to activate FGF signaling in cultured cells and to inhibit phosphate transport in proximal tubules perfused in vitro (13Baum M. Schiavi S. Dwarakanath V. Quigley R. Kidney Int. 2005; 68: 1148-1153Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). Klotho-deficient mice (Klotho–/– mice) and FGF23 deficient mice (Fgf23–/– mice) develop many common phenotypes, including shortened life span, growth retardation, infertility, muscle atrophy, hypoglycemia, and vascular calcification in the kidneys. Notably, they both have increased serum levels of phosphate (14Shimada T. Kakitani M. Yamazaki Y. Hasegawa H. Takeuchi Y. Fujita T. Fukumoto S. Tomizuka K. Yamashita T. J. Clin. Invest. 2004; 113: 561-568Crossref PubMed Scopus (1273) Google Scholar, 17Yoshida T. Fujimori T. Nabeshima Y. Endocrinology. 2002; 143: 683-689Crossref PubMed Scopus (169) Google Scholar). These observations have led us to the hypothesis that Klotho and FGF23 may function via a common signal transduction pathway. In this report we show that Klotho binds to multiple FGFRs and functions as a cofactor necessary for FGF signaling activation by FGF23. Expression Vectors—Complementary DNA containing the mouse FGFRs coding region (IMAGE Clone, Invitrogen, supplemental Fig. 1) were cloned into pcDNA3.1(+) expression vector (Invitrogen). Before subcloning, a V5-epitope tag was added to the C terminus and appropriate restriction enzyme sites to the both ends using synthetic oligonucleotides and polymerase chain reaction. Expression vectors for the mouse FGF23 resistant to proteolytic inactivation (R179Q) (18Shimada T. Muto T. Urakawa I. Yoneya T. Yamazaki Y. Okawa K. Takeuchi Y. Fujita T. Fukumoto S. Yamashita T. Endocrinology. 2002; 143: 3179-3182Crossref PubMed Scopus (384) Google Scholar), the transmembrane form of mouse Klotho, and the extracellular domain of mouse Klotho were cloned into pEF1 vector (Invitrogen) in the same way except that a FLAG-epitope tag was added to the C terminus. Cell Culture and Transfection—All cells except PC12 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and penicillin/streptomycin. PC12 cells were maintained in the same medium with additional 10% horse serum. Stable transformants of 293 cells expressing the full-length transmembrane form of Klotho (293KL) or the extracellular domain of Klotho (293KLΔTM) were isolated after selection with G418 (Invitrogen) for 14 days. Subconfluent 293, 293KL, and 293KLΔTM cells were transfected with the FGFR expression vector plasmids using the Lipofectamine transfection reagent (Invitrogen) according to the manufacturer's protocol. A CHO cell line that stably expresses the full-length Klotho (CHOKL) is a gift from Kyowa Hakko Kogyo Co. Ltd. (5Imura A. Iwano A. Tohyama O. Tsuji Y. Nozaki K. Hashimoto N. Fujimori T. Nabeshima Y. FEBS Lett. 2004; 565: 143-147Crossref PubMed Scopus (457) Google Scholar). Adenovirus Construction—A cDNA encoding the full-length transmembrane form of mouse Klotho with a C-terminal myc-eptope tag or green fluorescent protein (GFP) was inserted into the multiple cloning site of pShuttle-CMV (Qbiogene). After linearization, the shuttle vector was introduced into an electroporation-competent Escherichia coli BJ5183-AD-1 harboring the adenoviral backbone pAdEasy-1 (Stratagene). The recombinant vector was introduced into the adenovirus packaging cell line QBI-HEK293A (Qbiogene) using FuGENE 6 (Roche Applied Science). The viruses were amplified by several rounds of infection in QBI-HEK293A cells. Subconfluent HeLa cells or PC12 cells were infected with the adenovirus expressing Klotho or GFP (m.o.i. = 3 for HeLa and m.o.i. = 10 for PC12) 36 h before stimulation with FGF23 and then subjected to immunoblot analysis of FGF signaling pathway as described below. Immunoprecipitation and Immunoblotting—To prepare cell lysate, cells were snap-frozen in liquid nitrogen and lysed in the lysis buffer containing inhibitors for phosphatase and proteinase as described previously (2Kurosu H. Yamamoto M. Clark J.D. Pastor J.V. Nandi A. Gurnani P. McGuinness O.P. Chikuda H. Yamaguchi M. Kawaguchi H. Shimomura I. Takayama Y. Herz J. Kahn C.R. Rosenblatt K.P. Kuro-o M. Science. 2005; 309: 1829-1833Crossref PubMed Scopus (1397) Google Scholar). The lysate of 293KL or 293KLΔTM cells transfected with expression vectors for FGFRs was incubated with agarose beads conjugated with anti-V5 antibody (Sigma) or anti-FLAG antibody (Sigma) at 4 °C for 3 h. The beads were washed three times with Tris-buffered saline (TBS) containing 1% Triton X-100 (TBST) and three times with TBS. The washed beads were suspended in SDS-sample loading buffer and subjected to SDS-PAGE. The protein transferred to Hybond C Extra membrane (Amersham Biosciences) was incubated with anti-Klotho rat monoclonal antibody KM2119 (19Kato Y. Arakawa E. Kinoshita S. Shirai A. Furuya A. Yamano K. Nakamura K. Iida A. Anazawa H. Koh N. Iwano A. Imura A. Fujimori T. Kuro-o M. Hanai N. Takeshige K. Nabeshima Y. Biochem. Biophys. Res. Commun. 2000; 267: 597-602Crossref PubMed Scopus (132) Google Scholar) or anti-V5 antibody (Invitrogen) and then with horseradish peroxidase-linked secondary antibodies (Amersham Biosciences). The signals were detected with SuperSignal West Dura system (Pierce). For detecting Klotho binding to endogenous FGFRs in 293KL cells, cell lysate was immunoprecipitated with anti-FLAG-agarose in the same way as described above and then immunoblotted with antibodies against FGFR1 (Santa Cruz Biotechnology), FGFR2 (Santa Cruz Biotechnology), FGFR3 (Sigma), or KM2119. Preparation of Conditioned Medium Containing FGF23 (R179Q)—Serum-free conditioned medium was prepared by transfecting 293 cells with the mouse FGF23 (R179Q) expression vector. 293KL cells were stimulated with various doses of the conditioned medium and subjected to immunoblot analysis using anti-phospho-ERK antibody. The FGF23 activity in the conditioned medium was determined by comparing ERK phosphorylation with that induced by recombinant human FGF23 of known concentrations. The conditioned medium with the FGF23 activity equivalent to that of 10 ng/ml recombinant human FGF23 was used for the experiments. The same amount of serum-free conditioned medium from mock-transfected 293 cells was used as a negative control. Co-precipitation of Endogenous Klotho and FGFRs from Mouse Kidney— Kidney from a 129 mouse (200 mg) was homogenized in 2 ml of homogenizing buffer (20 mm HEPES, pH 7.4, 100 mm NaCl, 0.5 mm EDTA) containing protease inhibitors. The homogenate was incubated for 30 min at 4 °C after the addition of Triton X-100 (final 1%) and then centrifuged for 12 min at 18,000 × g two times to remove debris. The supernatant was precleared with 40 μl of protein A-Sepharose (Amersham Biosciences) conjugated with 20 μg of normal rabbit IgG for 2.5 h at 4 °C. The precleared lysate was incubated with 20 μl of protein A-Sepharose conjugated with 16 μg of anti-FGFR1 antibody or normal rabbit IgG for 2.5 h at 4 °C. The beads were processed in the same way as described above for immunoblot analysis using KM2119 and anti-FGFR1 antibody. The lysate of the kidney immunoprecipitated with anti-Klotho antibody KL11-A (Alpha Diagnostic International) was used as a positive control for Klotho. The mouse FGFR1c with a V5-epitope tag expressed in 293 cells was used as a positive control for FGFR1. FGF23 Pull-down Experiments—Lysate of 293 cells or 293KL cells transfected with the FGFR expression vectors was applied to anti-V5-agarose at 4 °C for 3 h. Serum-free conditioned medium of 293 cells or 293KLΔTM cells was applied to anti-FLAG-agarose at 4 °C for 3 h. The beads were washed four times with TBST and then incubated with conditioned medium of 293 cells transfected with the mouse FGF23 (R179Q) expression vector supplemented with 0.5% Triton X-100 at 4 °C for 3 h. The beads were washed three times with Krebs-Ringer-HEPES buffer containing 1% Triton X-100 and then three times with the same buffer without Triton X-100. The washed beads were suspended in SDS-sample loading buffer and subjected to immunoblot analysis using anti-V5 antibody, KM2119, or anti-FGF23 antibody (R PubMed Scopus Google Scholar). transfected 293KL cells that stably expressed the full-length transmembrane form of Klotho with various FGFR expression vectors and of the FGFRs be immunoprecipitated with Klotho. Klotho to FGFR However, in the ability of Klotho to FGFRs was observed the 1) were with Klotho than The and is in the C-terminal of the domain and known to binding affinity to as D.M. J. F. G. M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). FGFR2 was with Klotho than and Thus, Klotho may bind to multiple FGFRs with To test whether binding of Klotho to FGFRs may FGFRs and the ability of FGFRs to FGF23 was in the and of Klotho. FGF23 was with and in the of Klotho the possibility that FGF23 directly bind to Klotho. However, the extracellular domain of Klotho to FGF23 these These observations that FGF23 show with the Klotho-FGFR complex than with Klotho or FGFR alone. Klotho increased binding of FGF23 to it the ability of FGF23 to activate FGF signaling. To test this we stimulated 293KL cells or 293 cells with various of recombinant human FGF23 and phosphorylation of FGF receptor and MAP kinase 3 FGF23 to induce phosphorylation of and in 293 cells. In (10 FGF23 activated and in 293KL cells that Klotho enhanced the to FGF23 times without the of heparin or The similar were using two 293KL not In addition, we observed and ERK phosphorylation in 293KLΔTM cells the levels were to those in 293KL cells. Consistent with these findings, endogenous FGFRs in 293KL cells were with the full-length transmembrane Klotho to a with the extracellular domain of Klotho also that the extracellular domain of Klotho to expressed FGFRs in the same as the full-length Klotho Fig. on these we that both the full-length Klotho and the extracellular domain of Klotho function as cofactors necessary for activation of FGF signaling by FGF23 The function of FGF23 is to phosphate reabsorption in the renal tubular cells. endogenous Klotho and FGFR was observed in the mouse kidney Fig. To test whether the activity of Klotho to FGF23 be observed in cells as we infected PC12 and HeLa cells, which from and with adenovirus expressing the full-length Klotho and then stimulated with FGF23. In addition, we stimulated transformants of CHO cells expressing the full-length Klotho with FGF23. These cells the ability to to FGF23 Klotho was expressed Klotho binds to multiple it may the activity of than FGF23. stimulated 293 or 293KL cells with various doses of acidic and basic FGF and that Klotho not ability to activate FGF signaling basic FGF and supplemental Fig. The that FGF23 requires Klotho to activate FGF signaling may mice develop the observed in mice. However, mice show many not described in including arteriosclerosis, calcification in skin atrophy, and pulmonary emphysema (1Kuro-o M. Matsumura Y. Aizawa H. Kawaguchi H. Suga T. Utsugi T. Ohyama Y. Kurabayashi M. Kaname T. Kume E. Iwasaki H. Iida A. Shiraki-Iida T. Nishikawa S. Nagai R. Nabeshima Y. Nature. 1997; 390: 45-51Crossref PubMed Scopus (2811) Google Scholar). may that Klotho the activity of multiple binding to multiple the of Klotho on acidic and basic FGF was with its on FGF23 Fig. and may a to of of Klotho on the FGF23. In the of Klotho, FGF23 requires heparin or glycosaminoglycan as a cofactor to FGF signaling (13Baum M. Schiavi S. Dwarakanath V. Quigley R. Kidney Int. 2005; 68: 1148-1153Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar, X. Ibrahimi O.A. Goetz R. Zhang F. Davis S.I. Garringer H.J. Linhardt R.J. Ornitz D.M. Mohammadi M. White K.E. Endocrinology. 2005; 146: 4647-4656Crossref PubMed Scopus (177) Google Scholar, 16Yamashita T. Konishi M. Miyake A. Inui K. Itoh N. J. Biol. Chem. 2002; 277: 28265-28270Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar), that the of are for the activity of FGF23. Klotho is a and detected as two by immunoblot analysis which (5Imura A. Iwano A. Tohyama O. Tsuji Y. Nozaki K. Hashimoto N. Fujimori T. Nabeshima Y. FEBS Lett. 2004; 565: 143-147Crossref PubMed Scopus (457) Google Scholar). that the of Klotho was Klotho was with the high affinity binding such as and that a on the of Klotho may with these FGFRs. binding of FGFRs to the of Klotho was with the ability of the Klotho-FGFR complex to FGF23 is that a on Klotho protein may be involved in the high affinity FGFRs and FGF23. also that 293KLΔTM cells predominantly expressed the Fig. which partly 293KLΔTM cells to FGF23 than 293KL cells The that the extracellular domain of Klotho to FGF23 has the possibility that it may function as a factor in the kidney. Klotho is expressed in the tubules (1Kuro-o M. Matsumura Y. Aizawa H. Kawaguchi H. Suga T. Utsugi T. Ohyama Y. Kurabayashi M. Kaname T. Kume E. Iwasaki H. Iida A. Shiraki-Iida T. Nishikawa S. Nagai R. Nabeshima Y. Nature. 1997; 390: 45-51Crossref PubMed Scopus (2811) Google Scholar) and the extracellular domain of Klotho is shed and secreted (2Kurosu H. Yamamoto M. Clark J.D. Pastor J.V. Nandi A. Gurnani P. McGuinness O.P. Chikuda H. Yamaguchi M. Kawaguchi H. Shimomura I. Takayama Y. Herz J. Kahn C.R. Rosenblatt K.P. Kuro-o M. Science. 2005; 309: 1829-1833Crossref PubMed Scopus (1397) Google Scholar, 5Imura A. Iwano A. Tohyama O. Tsuji Y. Nozaki K. Hashimoto N. Fujimori T. Nabeshima Y. FEBS Lett. 2004; 565: 143-147Crossref PubMed Scopus (457) Google Scholar), it may on proximal tubules and with FGF23 to inhibit phosphate remains to be determined whether extracellular Klotho could function as a an factor in the of FGF23 signaling. was that mice defective in a protein that Klotho, increased and of S. Fujimori T. Furuya A. Satoh J. Nabeshima Y. Nabeshima Y. J. Clin. Invest. 2005; PubMed Scopus Google Scholar). these are with those observed in mice F. M. M. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, T. M. A. G. B. Cell Metab. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). The Klotho gene may have in the of FGF signaling. on Klotho are to of the complex FGF signaling system and its to with